Cellulose and hemicellulose constitute an important renewable and inexpensive carbon source for the production of fermentable sugars. Cellulose, consists of D-glucose units linked together in linear chains via β-1,4 glycosidic bonds. Hemicellulose consists primarily of a linear xylan backbone comprising D-xylose units linked together in via β-1,4 glycosidic bonds and numerous side chains linked to the xylose units via β-1,2 or β-1,3 glycosidic or ester bonds (e.g., L-arabinose, acetic acid, ferulic acid, etc).
Trichoderma reesei (the asexual anamorph of Hypocrea jecorina) is a filamentous fungus capable of producing a cellulase mixture comprising variety of cellulases and hemicellulases. These include two cellobiohydrolases, eight endoglucanases, four xylanases, two α-L-arabinofuranosidases, and a beta-mannanase. T. reesei also produces a number of accessory enzymes that assist in the generation of monosaccharides from the cellulose and hemicellulose, including acetyl xylan esterase, beta-xylosidase and several beta-glucosidases.
The regulation of the production of cellulases and hemicellulases by T. reesei is complex and controlled primarily at the transcriptional level in response to available carbon sources. Glucose represses cellulase gene expression through the action of transcriptional regulators such as cre1 (Strauss, J., et al., 1995, FEBS Letters 376: 103-107) and ace1 (Aro, N., et al., 2003, Appl. Environ. Microbiol. 69: 56-65). Under glucose-limiting conditions, cellulase transcription is derepressed, with full activation of transcription requiring the presence of an inducing carbohydrate, such as cellulose, or β-linked disaccharides such as cellobiose, sophorose, gentiobiose and lactose (Ilmen, M., et al., 1997, Appl. Environ. Microbiol. 63: 1298-1306). Cellulase-inducing carbohydrates (CIC) also lead to the activation of hemicellulase transcription (Mach, R. L. and Zeilinger, S., 2003, Appl. Microbiol. Biotechnol. 60: 515-522; Margolles-Clark et al., 1997, J. Biotechnol 57: 167-179). The xyr1 gene product has been shown to participate in the transcriptional activation of both hemicellulase and cellulase genes by xylose (Stricker, A. R., et al., 2006, Eukaryotic Cell 5: 2128-2137).
Although T. reesei produces low levels of xylanase activity under cellulase-inducing conditions, the enzyme system produced by cultures of T. reesei growing on xylose or other hemicellulose-derived carbohydrates is enriched in hemicellulase activities relative to cellulase activities. Production of secreted xylanase is enhanced by xylan (Bailey, M. J., et al., 1993, Appl. Microbiol. Biotechnol. 40: 224-229) and arabinose (Xiong et al., 2004, Appl. Microbiol. Biotech 64: 353-358). Transcription of hemicellulase genes is activated further by hemicellulose or its breakdown products as well as by cellulose. In Trichoderma reesei, transcription of the genes encoding xylanase 1 and 2 (xln1 and xln2) is activated by cellulose, sophorose, xylan and arabinitol, and transcription of arabinofuranosidase gene abf1 is activated by arabinose, arabinitol and xylan (Margolles-Clark, et al., 1997, J. Biotechnol. 57: 167-179). However, as a result of increased xylanase expression, the relative proportion of cellulase in the secreted enzyme composition is reduced. This results in decreased specific activity of the cellulase and, as a consequence, higher dosages of total protein are needed for effective hydrolysis of cellulosic substrates.
Provision of equimolar amounts of cellobiose and xylobiose results in similar levels of xylanase activity in shake-flask batch cultures of T. reesei strain QM9414 (Zeilinger, S., et al., 1996, J. Biol. Chem. 271: 25624-25629). The cellulase activities produced by the two cultures were not reported. The relative proportion of xylanase secreted by T. reesei strain RutC30 in fed-batch culture was increased from 0.8% to 3.5% by changing the carbon source from 100% lactose to 75% lactose/25% xylose (Margeot, A., et al., poster presentation at 29th Symposium on Biotechnology for Fuels and Chemical, Denver, Colo., USA). At the same time, the combined proportion of the four major cellulase components (cellobiohydrolases 1 and 2, endoglucanases 1 and 2) was reduced from 82% to 62%. The proportion of other hemicellulases and cellulases was not reported. Shake-flask batch cultures of T. reesei strain RutC30, using saw-dust hydroysates, were reported to produce similar levels of secreted cellulase activity (in terms of filter paper units per ml of culture) as cellulose hydrolysates (Lo, C -H. et al., 2005, Appl. Biochem. Biotechnol. Spring (121-124): 561-573). The saw-dust hydrolysates were produced by treatment with concentrated sulfuric acid and, in the case of saw dust, consisted of 25-40% hemicellulose-derived carbohydrates, 5-9% cellulase-inducing carbohydrates, 13-45% glucose and 2-45% oligosaccharides. However, as no information is given concerning the effect of the sawdust hydrolysates on hemicellulase activity or total secreted protein, the impact of the sawdust hydrolysates on the relative proportions of cellulose and hemicellulase in the secreted protein could not be estimated.
The relative proportion of hemicellulases can also be manipulated by adjusting the pH of the fermentation culture medium (Bailey, M. J., et al., 1993, Appl. Microbiol. Biotechnol. 40: 224-229); the proportion of xylanase activity relative to cellulase activity is enhanced at pH 6-7. The transcription of xylanase genes in Aspergillus are subject to regulation by pH-dependent transcriptional regulator pacC (Maccabe, A. P., et al., 1998, J. Bacteriol. 180:1331-1333).
There are situations in which it is desirable to produce cellulase mixtures with a high proportion of cellulases from fungal cultures using carbohydrate sources comprising mainly xylose and other pentose sugars derived from hemicellulose, such as those produced by chemical treatments of lignocellulosic biomass. Methods for the pretreatment of hemicellulose-containing lignocellulosic biomass are described in U.S. Pat. Nos. 4,461,648, 5,916,780, 6,090,595, 6,043,392, 4,600,590; Weil et al., 1997, Appl. Biochem. Biotechnol. 681: 21-40; and Öhgren, K., et al., 2005, Appl. Biochem. Biotechnol. Spring (121-124): 1055-1067 (which are incorporated herein by reference).